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A novel series of c-jun N-terminal kinase (JNK) inhibitors were designed and developed from a high-throughput-screening hit. Through the optimization of the piperazine amide 1, several potent compounds were discovered. The X-ray crystal structure of 4g showed a unique binding mode different from other well known JNK3 inhibitors.
The c-jun N-terminal kinase (JNKs) are serine/threonine protein kinases and members of the mitogen-activated protein kinase (MAPK) family which can be activated in response to various stimuli such as environmental stress, cytokines and fatty acids.1 Activated JNK phosphorylates both cytoplasmic substrates (cytoskeletal proteins, Bcl-2, APP) as well as nuclear transcription factors (c-jun, ATF-2) which can lead to an array of signal transduction cascades including cell death, cell survival and growth. Three JNK isoforms (JNK1, 2, and 3) have been identified, with JNK1 and JNK2 widely expressed in all tissues, whereas JNK3 is selectively expressed in the brain, heart, and testis.2,3
In recent studies, JNK-1, often in concert with JNK-2 has been suggested to play a central role in the development of obesity-induced insulin resistance which implies therapeutic inhibition of JNK1 may provide a potential solution in type-2 diabetes mellitus.4,5 JNK2 has been described in the pathology of autoimmune disorders such as rheumatoid arthritis and asthma, and it also has been implicated to play a role in cancer, as well as in a broad range of diseases with an inflammatory component.6-10 JNK3 has been shown to mediate neuronal apoptosis which makes inhibiting this isoform a promising therapeutic target for neurodegenerative diseases such as Parkinson's disease, Alzheimer's disease and other CNS disorders.11-13 Therefore, developing JNK inhibitors as therapeutics has gained considerable interest over the past few years.14-21
In this Letter we report a novel series of pan-JNK inhibitors identified in a high-throughput-screening campaign of our in-house sample collection. Compound 1 was identified as an ATP-competitive pan-JNK inhibitor with IC50 values 1.0 μM and 0.49 μM versus JNK3 and JNK1, respectively, with no inhibition against p38 (>20 μM). Encouraged by the lead candidates structural simplicity and by its promising degree of selectivity against p38, we initiated lead optimization. Herein we report the synthesis, characterization, and SAR (Structure-Activity-Relationships) of piperazine 1 (see Fig. 1).
The synthesis of analogs of 1 are outlined in Scheme 1. A 1,2-disubstituted-3-nitrobenzene (2) was treated with a piperazine or piperidine in toluene and refluxed overnight. The resulting nitrobenzene was reduced to the aniline (3) via hydrogenation or SnCl2 and then treated with the various acid chlorides in pyridine to give R1-derivatized compounds (Tables 1--3,3, 4, 6, and 9). Further SAR of the piperazine was accomplished by BOC-cleavage and funtionalization to afford analogs of 4 (Table 1).
Compared to the lead structure (1), the unprotected piperazine (4a) was a 10-fold less potent JNK3 inhibitor while simple alkyl substitutions (4b-d) provided little change in potency (Table 1). However, small side chains which contain unsaturation or sp-2 character seemed to provide a small boost in potency (up to six-fold) (4e-i). Its unclear if this was the result of a favorable pi-stacking interaction that was no longer available to the sterically larger benzyl or phenethyl analogs. Direct arylation (4l) or acylation of the piperazine provided no inhibition advantage (4m-o). As for varying the 3-substituent on the phenyl ring, it seemed like small groups were tolerated (Cl, F, Me), but larger groups led to loss of activity (4r-u). Like lead hit 1, all analogs showed no selectivity between JNK3 and JNK1, and if anything, were slightly more potent against JNK1. All compounds also showed no inhibition against p38 (data not shown).
SAR of the aniline amide group proved to be equally frustrating (Table 2). We desired to find a replacement for the electron rich furan group with something that might be more metabolically stable, however attempts to modify the 5-bromofuran moiety and maintain potency were difficult.23 Of all the substitutions examined, the only sub-micromolar inhibitor was 5-bromothiazole 6b. All others led to substantial losses in potency. Once again, the nature of the interaction, and the special requirement for the bromofuran or thiazole to maintain activity was unclear.
Given the finding that 3-methyl substitution was slightly more potency enhancing than 3-chloro (4p vs 4e), future SAR was conducted on the 3-methylsubstituted analogs (Table 3). Additionally, a spiroketal-piperidine was found to be more potency enhancing than the terminally substituted piperazine group. While attempts to replace the bromofuran ring proved challenging, we were able to replace the bromine atom without complete loss of activity. The 5-chlorofuran was the most promising substitution with little affect on potency, however all other substitutions tried, led to a 5- 10-fold drop. This drop in potency did not appear to be a size-related phenomena given that both smaller and larger groups are equally less active.
A few compounds from this series were further profiled in a cell-based assay (4f IC50 = 3.3 μM; 4d IC50 = 2.1 μM) and showed a considerable shift in activity (30-40-fold).24 Whether the shift arises from the higher ATP concentration in cells or from lack of cell penetration is still under investigation, but this is not an uncommon observation.25
To help explain the binding mode of inhibitor to enzyme and to aid in the design of more potent analogs, an X-ray crystal structure of a compound from this class bound to JNK3 was pursued. Fortunately, 4g provided crystals suitable for X-ray diffraction and the interactions between ligand and enzyme are shown in Figure 2.26 The inhibitor occupied the ATP binding site, with the bromofuran ring placed deep within the adenosine binding region. The furan was up against the gatekeeper Met 146, but interacting only through van der Waals contacts. The piperazine also formed van der Waals contacts with Ile 70 and Val 78 and to a lesser extent it is interacting in a similar fashion with Val 196 and Leu 206. These are important because they help stabilize the p-loop. The amide oxygen atom in 4g bound to the backbone hinge (Met 149) in JNK3 and was also close enough to form an electrostatic interaction with the carboxy main chain oxygen of Glu 147. This binding mode is not unlike that described for other structurally unique JNK inhibitors in the literature which benefit not from a strong bidentate interaction with the hinge region of the enzyme, but from the sum of many weak interactions.27 It may also help to explain why small perturbations in the molecular structure greatly affect inhibition.
In summary, a novel class of piperazine amides were developed from the high-throughput-screening lead 1. Lead compound optimization produced a series of potent pan-JNK inhibitors inactive against p38 that displayed moderate activity in a JNK cell-based assay. An X-ray crystal structure with JNK3 revealed an unusual binding mode which may be helpful in designing more potent analogs. Synthesis and characterization of these compounds is in progress and will be reported in due course.
We would like to thank Peter Hodder and the Scripps Florida HTS Group for screening as well as Professor Bill Roush for helpful discussions. We also thank the beamline staff at SER-CAT (APS 22-ID) for the data collection help and support in solving the protein structure. This work was supported by NIH grant U01 NS057153 awarded to P.L.